Enhanced ROCK1 dependent contractility in fibroblast from chronic obstructive pulmonary disease patients

Background: During wound healing processes fibroblasts account for wound closure by adopting a contractile phenotype. One disease manifestation of COPD is emphysema which is characterized by destruction of alveolar walls and our hypothesis is that fibroblasts in the COPD lungs differentiate into a more contractile phenotype as a response to the deteriorating environment. Methods: Bronchial (central) and parenchymal (distal) fibroblasts were isolated from lung explants from COPD patients (n = 9) (GOLD stage IV) and from biopsies from control subjects and from donor lungs (n = 12). Tissue-derived fibroblasts were assessed for expression of proteins involved in fibroblast contraction by western blotting whereas contraction capacity was measured in three-dimensional collagen gels. Results: The basal expression of rho-associated coiled-coil protein kinase 1 (ROCK1) was increased in both centrally and distally derived fibroblasts from COPD patients compared to fibroblasts from control subjects (p < 0.001) and (p < 0.01), respectively. Distally derived fibroblasts from COPD patients had increased contractile capacity compared to control fibroblasts (p < 0.01). The contraction was dependent on ROCK1 activity as the ROCK inhibitor Y27632 dose-dependently blocked contraction in fibroblasts from COPD patients. ROCK1-positive fibroblasts were also identified by immunohistochemistry in the alveolar parenchyma in lung tissue sections from COPD patients. Conclusions: Distally derived fibroblasts from COPD patients have an enhanced contractile phenotype that is dependent on ROCK1 activity. This feature may be of importance for the elastic dynamics of small airways and the parenchyma in late stages of COPD

The Hidden Story of Heterogeneous B-raf V600E Mutation Quantitative Protein Expression in Metastatic Melanoma-Association with Clinical Outcome and Tumor Phenotypes

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter-and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma

The Human Melanoma Proteome Atlas—Complementing the melanoma transcriptome

The MM500 meta‐study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. Somatic mutations and their effect on the transcriptome have been extensively characterized in melanoma. However, the effects of these genetic changes on the proteomic landscape and the impact on cellular processes in melanoma remain poorly understood. In this study, the quantitative mass‐spectrometry‐based proteomic analysis is interfaced with pathological tumor characterization, and associated with clinical data. The melanoma proteome landscape, obtained by the analysis of 505 well‐annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. More than 50 million MS/MS spectra were analyzed, resulting in approximately 13,6 million peptide spectrum matches (PSMs). Altogether 13 176 protein‐coding genes, represented by 366 172 peptides, in addition to 52 000 phosphorylation sites, and 4 400 acetylation sites were successfully annotated. This data covers 65% and 74% of the predicted and identified human proteome, respectively. A high degree of correlation (Pearson, up to 0.54) with the melanoma transcriptome of the TCGA repository, with an overlap of 12 751 gene products, was found. Mapping of the expressed proteins with quantitation, spatiotemporal localization, mutations, splice isoforms, and PTM variants was proven not to be predicted by genome sequencing alone. The melanoma tumor molecular map was complemented by analysis of blood protein expression, including data on proteins regulated after immunotherapy. By adding these key proteomic pillars, the MM500 study expands the knowledge on melanoma disease

Correlation of histopathologic characteristics to protein expression and function in malignant melanoma

BACKGROUND: Metastatic melanoma is still one of the most prevalent skin cancers, which upon progression has neither a prognostic marker nor a specific and lasting treatment. Proteomic analysis is a versatile approach with high throughput data and results that can be used for characterizing tissue samples. However, such analysis is hampered by the complexity of the disease, heterogeneity of patients, tumors, and samples themselves. With the long term aim of quest for better diagnostics biomarkers, as well as predictive and prognostic markers, we focused on relating high resolution proteomics data to careful histopathological evaluation of the tumor samples and patient survival information. PATIENTS AND METHODS: Regional lymph node metastases obtained from ten patients with metastatic melanoma (stage III) were analyzed by histopathology and proteomics using mass spectrometry. Out of the ten patients, six had clinical follow-up data. The protein deep mining mass spectrometry data was related to the histopathology tumor tissue sections adjacent to the area used for deep-mining. Clinical follow-up data provided information on disease progression which could be linked to protein expression aiming to identify tissue-based specific protein markers for metastatic melanoma and prognostic factors for prediction of progression of stage III disease. RESULTS: In this feasibility study, several proteins were identified that positively correlated to tumor tissue content including IF6, ARF4, MUC18, UBC12, CSPG4, PCNA, PMEL and MAGD2. The study also identified MYC, HNF4A and TGFB1 as top upstream regulators correlating to tumor tissue content. Other proteins were inversely correlated to tumor tissue content, the most significant being; TENX, EHD2, ZA2G, AOC3, FETUA and THRB. A number of proteins were significantly related to clinical outcome, among these, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in progression from stage III to stage IV disease and poor survival. CONCLUSION: In this feasibility study, promising results show the feasibility of relating proteomics to histopathology and clinical outcome, and insight thus can be gained into the molecular processes driving the disease. The combined analysis of histological features including the sample cellular composition with protein expression of each metastasis enabled the identification of novel, differentially expressed proteins. Further studies are necessary to determine whether these putative biomarkers can be utilized in diagnostics and prognostic prediction of metastatic melanoma

HDAC2-independent anti-inflammatory effects of budesonide in human lung fibroblasts

Background: Reduced response of COPD patients to the anti-inflammatory therapy with glucocorticoids may be due to reduced expression of histone deacetylase (HDAC) in alveolar macrophages and bronchial epithelial cells as suggested by recent studies. Lung fibroblasts release inflammatory mediators and are key cells in tissue remodeling following airway inflammation. However, HDAC expression and its role in mediating glucocorticoid effect on fibroblast functions have not been studied. Methods and results: Human fetal lung fibroblasts (HFL-1) were exposed to IL-1β +TNF-α (1ng/ml each), which stimulated release of IL-6, IL-8 and metalloproteinases MMP-1 and MMP-3. These responses were inhibited by the glucocorticoid budesonide (0.1-100nM) in a concentration-dependent manner. An HDAC inhibitor (trichostatin A) did not reverse the effects of budesonide on release of cytokines and MMPs in HFL-1 cells while it blocked the inhibitory effects of budesonide in human bronchial epithelial cells and monocytes. Furthermore, siRNA targeting HDAC2 did not interfere with the inhibitory effects of budesonide on HFL-1 MMP release. Exposure to cigarette smoke extract (5%) did not affect HDAC2 protein expression in HFL-1 cells and did not interfere with the budesonide effects. Finally, there was no statistically significant difference between COPD and control subjects in HDAC2 expression and the effects of budesonide on cytokine or MMP release from lung fibroblasts. Conclusions: HDAC2 is not required for budesonide to inhibit MMP and cytokine release by lung fibroblasts. These results also suggest that budesonide has a potential to counteract fibroblast-mediated tissue remodeling following airway inflammation in COPD

Cigarette smoke extract modulates respiratory defence mechanisms through effects on T-cells and airway epithelial cells.

Chronic obstructive pulmonary disease (CCPD) is a disease primarily caused by cigarette smoking, which in turn has been shown to affect the susceptibility to and progression of airway infections. The question addressed in this study was how components from cigarette smoke could affect the defence mechanisms of T-cells and epithelial cells, and thereby contribute to the development of the COPD pathology. T-cells and monocytes were isolated from buffycoats from healthy donors and T-cell responses studied in response to cigarette smoke extract (CSE). Activation level (CD25 expression), proliferation (BrdU incorporation) and intracellular expression of the cytotoxic markers granzyme-b and TIA-1 were determined using flowcytometry. Normal human bronchial epithelial cells were obtained from Cambrex and differentiated in air-liquid interface cultures. After exposure to CSE barrier function (trans-epithelial electric resistance, TEER), MUC5AC and interleukin-8 production were measured. T-cell activation, proliferation and expression of the cytotoxic proteins granzyme-b and TIA-1 were significantly reduced in response to 0.5-1% of CSE. The epithelial cells were more resistant to CSE and responded at doses 20 times higher than T-cells. The expression of interteukin-8 and MUC5AC was significantly increased after exposure to 15% and 30% CSE and TEER was largely unaffected at 30% CSE but clearly reduced at 40% CSE. This study shows that mechanisms, in both T-cells and airway epithelial cells, involved in the defence against infectious agents are modulated by CSE. (c) 2005 Elsevier Ltd. All rights reserved

Workflow for large-scale analysis of melanoma tissue samples

AbstractThe aim of the present study was to create an optimal workflow for analysing a large cohort of malignant melanoma tissue samples. Samples were lysed with urea and enzymatically digested with trypsin or trypsin/Lys C. Buffer exchange or dilution was used to reduce urea concentration prior to digestion. The tissue digests were analysed directly or following strong cation exchange (SCX) fractionation by nano LC–MS/MS. The approach which resulted in the largest number of protein IDs involved a buffer exchange step before enzymatic digestion with trypsin and chromatographic separation in 120min gradient followed by SCX–RP separation of peptides

Biomarkers of early chronic obstructive pulmonary disease (COPD) in smokers and former smokers. Protocol of a longitudinal study.

Chronic obstructive pulmonary disease (COPD) is an irreversible disease, diagnosed predominantly in smokers. COPD is currently the third leading cause of death worldwide. Far more than 15 % of smokers get COPD: in fact, most develop some amount of pulmonary impairment. Smoking-related COPD is associated with both acute exacerbations and is closely correlated to comorbidities, such as cardiovascular disease and lung cancer. The objective of our study (KOL-Örestad) is to identify biomarkers in smokers and ex-smokers, with early signs of COPD, and compare these biomarkers with those of non-smokers and healthy smokers/ex-smokers. The participants in the study are recruited from Örestadskliniken, a primary health care clinic in Malmö, Sweden

Effect of budesonide on fibroblast-mediated collagen gel contraction and degradation

Background: The balance between production and degradation of extracellular matrix is crucial in maintaining normal tissue structure. Budesonide is known to have anti-inflammatory effects, but its effect on fibroblast-mediated tissue repair and remodeling has not been fully studied. Methods: Using human fetal lung fibroblasts (HFL-1 cells) in a three-dimensional collagen gel culture system, the current study investigated the effect of budesonide (1-1000 nM) on collagen gel contraction and degradation in the presence or absence of inflammatory cytokines (IL-1β and TNF-α; 5 ng/ml each) and, in order to activate latent proteases, the serine protease trypsin (0.25 μg/ml). Results: Inflammatory cytokines significantly inhibited collagen gel contraction mediated by lung fibroblasts. Budesonide counteracted the effect of cytokines in a concentration-dependent manner (to 50%, p<0.01). Budesonide (100 nM) almost completely inhibited the release and mRNA expression of metalloproteinases (MMP) -1, -3 and -9 induced by the cytokines (p<0.05). Exposure to the cytokines plus trypsin increased collagen degradation and activation of the MMPs. Budesonide blocked both the enhanced collagen degradation (p<0.01) and suppressed the trypsin-mediated activation of cytokine-induced MMP-9 and MMP-3. Similar effects were observed with dexamethasone (1 μM), suggesting a class effect. Conclusions: These findings suggest that budesonide directly modulates contraction of collagen and can decrease collagen degradation under inflammatory conditions through suppressing release and activation of MMPs. By modulating the release and activity of MMPs, inhaled budesonide may be able to modify airway tissue repair and remodeling